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. 2010 Sep 30;5(9):e13087.
doi: 10.1371/journal.pone.0013087.

Decreased Fat Storage by Lactobacillus Paracasei Is Associated With Increased Levels of Angiopoietin-Like 4 Protein (ANGPTL4)

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Free PMC article

Decreased Fat Storage by Lactobacillus Paracasei Is Associated With Increased Levels of Angiopoietin-Like 4 Protein (ANGPTL4)

Linda Aronsson et al. PLoS One. .
Free PMC article

Abstract

Background: Intervention strategies for obesity are global issues that require immediate attention. One approach is to exploit the growing consensus that beneficial gut microbiota could be of use in intervention regimes. Our objective was to determine the mechanism by which the probiotic bacteria Lactobacillus paracasei ssp paracasei F19 (F19) could alter fat storage. Angiopoietin-like 4 (ANGPTL4) is a circulating lipoprotein lipase (LPL) inhibitor that controls triglyceride deposition into adipocytes and has been reported to be regulated by gut microbes.

Methodology/principal findings: A diet intervention study of mice fed high-fat chow supplemented with F19 was carried out to study potential mechanistic effects on fat storage. Mice given F19 displayed significantly less body fat, as assessed by magnetic resonance imaging, and a changed lipoprotein profile. Given that previous studies on fat storage have identified ANGPTL4 as an effector, we also investigated circulating levels of ANGPTL4, which proved to be higher in the F19-treated group. This increase, together with total body fat and triglyceride levels told a story of inhibited LPL action through ANGPTL4 leading to decreased fat storage. Co-culture experiments of colonic cell lines and F19 were set up in order to monitor any ensuing alterations in ANGPTL4 expression by qPCR. We observed that potentially secreted factors from F19 can induce ANGPTL4 gene expression, acting in part through the peroxisome proliferator activated receptors alpha and gamma. To prove validity of in vitro findings, germ-free mice were monocolonized with F19. Here we again found changes in serum triglycerides as well as ANGPTL4 in response to F19.

Conclusions/significance: Our results provide an interesting mechanism whereby modifying ANGPTL4, a central player in fat storage regulation, through manipulating gut flora could be an important gateway upon which intervention trials of weight management can be based.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. F19 supplementation decreases fat storage in vivo.
A) The two groups (5 mice/group) of high-fat (HF) and high-fat supplemented with F19 (HF+F19) were pair-fed, referenced here by food consumption in grams/week. B) Free fatty acid content in the serum of the two groups. C) Lipoprotein profiles of both cholesterol and triglyceride contents of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). D) Western blot for full length ANGPTL4 in pooled serum (10 µg protein loaded) from HF and HF+F19 mice along with numerical representation of the same. E) Representative images from MRI visualizing fat depots (in white) in both the abdominal and visceral regions. F) Body fat percentages for the two groups. Stars represent P = 0.045 (*), P = 0.002 (**) using Student's t-test.
Figure 2
Figure 2. Probiotics upregulate ANGPTL4 expression in colonic cell lines.
A) Real-time PCR of ANGPTL4 mRNA in HCT116 cells co-cultured with Lactobacillus F19 (F19; 107/ml), Bifidobacterium lactis (BB12; 107/ml) and Bacteroides thetaiotaomicron (B.theta; 107/ml) respectively for 6 h were compared to non-treated (NT) control. B) Analysis of ANGPTL4 mRNA after 6 h stimulation with F19 at different concentrations in HCT116 cells. C) Time-course of F19 (108) on ANGPTL4 mRNA expression in HCT116 cells. D) Western (50 µg) of full length ANGPTL4 in HCT116 cells treated with F19 for 6 h and collected after 24 h. Actin is shown as loading control. E) ANGPTL4 mRNA in the colon carcinoma cell lines LoVo, HT29 and SW480. Real-time PCR data are presented as means with standard errors. All data are representative of at least 3 independent experiments.
Figure 3
Figure 3. ANGPTL4 mRNA expression is regulated by F19 secreted factors.
A) Real-time PCR of HCT116 cells stimulated for 6 h by live (F19) or heat-killed F19 (H-K F19) as well as fresh (CM) or heat-inactivated conditioned media of F19 (H-I CM) compared to non-treated (NT) control. B) Comparison between 6 h stimulation with conditioned media (CM) and F19 culture supernatant (CS) on ANGPTL4 expression. Bars signify means with standard errors. Results are representative of at least 3 independent experiments, and stars represent P<0.02 (*), P<0.01 (**), P<0.001 (***) (Student's t-test).
Figure 4
Figure 4. F19 mediated induction of ANGPTL4 gene expression may be mediated through PPARs.
A) Stimulation of ANGPTL4 gene expression using the PPARα ligand WY-14643 (WY), PPARγ ligand Rosiglitazone (Rosi), and PPARδ ligand GW0742 (G07). B) Inhibition of ANGPTL4 response in HT29 cells after 6 h incubation with F19 by siRNA for PPARα, PPARγ, and PPARδ. Controls for siRNA efficiency of each inhibition are included as separate qPCR graphs. Expression data are presented with standard errors of the mean. Data are representative of at least 3 independent experiments, and stars represent P<0.05 (*), P<0.01 (**), P<0.001 (***) (Student's t-test).
Figure 5
Figure 5. F19 monocolonization in germ-free mice increases ANGPTL4 protein levels in serum.
A) Western blot for full length ANGPTL4 levels in a serially diluted (50, 25, 12.5, 6.25 µg protein) serum pool of control (PBS) and mono-infected mice (F19), along with a collated numerical representation of the same western corrected for loading. B) Cholesterol and triglyceride profiles of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) in control (PBS) and mono-infected serum (F19). Indicated bars represent the average value of each data set, n = 6, while stars represent P = 0.045 (*), P = 0.002 (**) (Student's t-test).

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